Revision 3
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IF-IC

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

62

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q13501

Entrez-Gene Id:

8878

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunofluorescence (Immunocytochemistry) 1:50 - 1:200

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb recognizes endogenous levels of SQSTM1/p62 protein only when phosphorylated at Ser403.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser403 of human SQSTM1/p62 protein.

Background

Sequestosome 1 (SQSTM1, p62) is a ubiquitin binding protein involved in cell signaling, oxidative stress, and autophagy (1-4). It was first identified as a protein that binds to the SH2 domain of p56Lck (5) and independently found to interact with PKCζ (6,7). SQSTM1 was subsequently found to interact with ubiquitin, providing a scaffold for several signaling proteins and triggering degradation of proteins through the proteasome or lysosome (8). Interaction between SQSTM1 and TRAF6 leads to the K63-linked polyubiquitination of TRAF6 and subsequent activation of the NF-κB pathway (9). Protein aggregates formed by SQSTM1 can be degraded by the autophagosome (4,10,11). SQSTM1 binds autophagosomal membrane protein LC3/Atg8, bringing SQSTM1-containing protein aggregates to the autophagosome (12). Lysosomal degradation of autophagosomes leads to a decrease in SQSTM1 levels during autophagy; conversely, autophagy inhibitors stabilize SQSTM1 levels. Studies have demonstrated a link between SQSTM1 and oxidative stress. SQSTM1 interacts with KEAP1, which is a cytoplasmic inhibitor of NRF2, a key transcription factor involved in cellular responses to oxidative stress (3). Thus, accumulation of SQSTM1 can lead to an increase in NRF2 activity.
Phosphorylation of SQSTM1 at Ser403 increases its affinity for polyubquitinated chains, resulting in enhanced autophagic clearance (13,14). This site has been reported to be phosphorylated by casein kinase 2 (CK2), as well as by the innate immunity regulator TBK-1.

  1. Kirkin, V. et al. (2009) Mol Cell 34, 259-69.
  2. Seibenhener, M.L. et al. (2007) FEBS Lett 581, 175-9.
  3. Komatsu, M. et al. (2010) Nat Cell Biol 12, 213-23.
  4. Bjørkøy, G. et al. (2006) Autophagy 2, 138-9.
  5. Joung, I. et al. (1996) Proc Natl Acad Sci USA 93, 5991-5.
  6. Sanchez, P. et al. (1998) Mol Cell Biol 18, 3069-80.
  7. Puls, A. et al. (1997) Proc Natl Acad Sci USA 94, 6191-6.
  8. Vadlamudi, R.K. et al. (1996) J Biol Chem 271, 20235-7.
  9. Wooten, M.W. et al. (2005) J Biol Chem 280, 35625-9.
  10. Bjørkøy, G. et al. (2005) J Cell Biol 171, 603-14.
  11. Komatsu, M. et al. (2007) Cell 131, 1149-63.
  12. Pankiv, S. et al. (2007) J Biol Chem 282, 24131-45.
  13. Matsumoto, G. et al. (2011) Mol Cell 44, 279-89.
  14. Pilli, M. et al. (2012) Immunity 37, 223-34.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IF-IC: Immunofluorescence (Immunocytochemistry)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Revision 3
#39786

Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb

Western Blotting Image 1: Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing GFP-tagged human SQSTM1/p62 (hSQSTM1/p62-GFP; +) and human GST-tagged IKK-ε (hIKK-ε-GST; +) as indicated, using Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 2: Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa, C2C12, and H-4-II-E cells, untreated (-) or treated with Chloroquine #14774 (50 μM, overnight; +), using Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb (upper), total SQSTM1/p62 (D1Q5S) Rabbit mAb #39749 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 3: Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb Expand Image
Western blot analysis of STF-66247-treated 786-O cell extracts, that were either untreated (-) or treated with Lambda Phosphatase plus Calf Intestinal Phosphatse ( λPPase/CIP; +), using Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb (upper), total SQSTM1/p62 (D5L7G) Mouse mAb #88588 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 4: Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb Expand Image
Western blot analysis of extracts from 786-O cells, untreated (-) or treated with STF-66247 (1 μM, indicated times), using Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb (upper), total SQSTM1 (D5L7G) Mouse mAb #88588 (middle), or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Immunofluorescence Image 1: Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of 786-O cells, untreated (left) or treated with STF-66247 (1 μM, 18 hr; right), using Phospho-SQSTM1/p62 (Ser403) (D8D6T) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).